This work was supported by the grants from National Natural Science Foundation of China (31201086, 31470759 and 31671219) and Shanghai Natural Science Foundation (12ZR1436900).

C57BL/6 mice are bred in specific pathogen-free conditions. All animal experimental methods are in compliance with the guidelines approved by the Animal Care and Ethics Committee at Second Military Medical University.
1. Dissection of Postnatal Developing Pituitary Gland
<ol>
	<li>Perform anesthesia: Place neonatal mice (P0 - P5) on crushed ice to induce hypothermia. For mice older than 5 days of age, inject 5% urethane (30 &#181;L/g of body weight) intraperitoneally to induce anesthesia. Assess responses to tail/toe pinches. Proceed only after the mouse is unresponsive to the noxious stimuli.</li>
	<li>Secure the mouse in the supine position (lying on the back with face upward) by gently taping the forepaws and hindpaws to a work surface that is capable of being pinned (i.e., Styrofoam) inside a chemical fume hood.</li>
	<li>Perform transcardial perfusion.
	<ol>
		<li>Cut the mouse ribcage with surgical scissors to expose the heart (and other thoracic organs).</li>
		<li>Secure the beating heart with blunt forceps and insert a 26G (0.45 mm) needle into the left ventricle. The needle is attached to a 10 mL syringe filled with heparinized phosphate buffered saline (PBS; supplemented with 5 mg/mL sodium nitrite and 10 U/mL heparin, pH 7.4, warm to 37 &#176;C before use). Immediately cut the right atrium with fine scissors and begin to perfuse the body with the warm PBS until the fluid exiting the right atrium is clear. 
		NOTE: Approximate 5 - 10 mL PBS is needed for neonatal mice and 10 - 20 mL PBS for adult mice.</li>
		<li>Keep the needle in place and change to another syringe filled with 4% paraformaldehyde (PFA; pH 7.4). Perfuse 10 mL and 20 mL of fixative for neonatal and P21 mouse, respectively. 
		Caution: PFA is toxic. It may cause skin and respiratory irritations, and eye damage. Wear gloves and work under a chemical hood.</li>
	</ol>
	</li>
	<li>Decapitate the PFA-fixed mouse and cut the skull bone open with scissors.
	<ol>
		<li>Gently lift the hindbrain from the base of the skull with small forceps. At the first sign of the sella turcica, stop lifting but hold the hindbrain, cut the pituitary stalk and nerve fibers connecting to the base of the brain with fine scissors. This step is critical to ensure that the pituitary is not lifted away with the brain.</li>
		<li>Then keep lifting the brain and remove the whole brain to expose the pituitary gland fully. Cut the entire sella region including the pituitary gland, lateral trigeminal nerves, and the beneath sphenoid bone with scissors (approximately 3 x&#160;5 mm2).</li>
	</ol>
	</li>
	<li>Put the dissected tissue into a 35 mm dish or one well of a 6-well plate containing 2 mL cold 4% PFA (pH 7.4). Fix the tissue at 4 &#176;C for 40 min for P0 - P7 pituitaries, 1 h for P14 pituitaries, 1.5 h for P21 - P28 pituitaries, and 3 h for adult pituitaries.</li>
	<li>Further dissociation of pituitary
	<ol>
		<li>Wash the fixed tissue with 10 mL PBS, 5 changes, 15 min each. The neonatal pituitary gland together with its surrounding tissues and the beneath sphenoid bone can be processed directly to dehydration. However, pituitary glands from mice older than 5 days should be dissociated further under a stereo microscope.</li>
		<li>Put the fixed tissue into a 35 mm dish containing 1 mL PBS. For P5 - P14 pituitaries, remove the connective membranes between the nerves and the bone with fine forceps and scissors, and carefully isolate the pituitary gland together with lateral trigeminal nerves as a whole but leaving the sella turcica.The isolated gland and nerves are connected by connective membranes with a &#34;H&#34;-like appearance (Figure 1B).</li>
		<li>For P21 and adult pituitaries, remove the nerves and connective membranes around the pituitary and free the gland from the surrounding tissues. Wrap the pituitary tissue with lens cleaning tissue paper and put it into an embedding cassette. 
		NOTE: Wrapping the tiny pituitary with lens cleaning tissue paper helps to prevent any potential loss of specimens and preserve tissue integrity in the following paraffin-embedding procedure. It may not be necessary to wrap the pituitary if it is processed in a small bottle in the cryo-embedding procedure.</li>
	</ol>
	</li>
</ol>
2. Paraffin Embedding and Sectioning
<ol>
	<li>Dehydrate the specimens in embedding cassettes with 50%, 65%, 75%, and 95% ethanol solutions for 15 min each. Subsequently dehydrate with 3 changes of pure ethanol, 3 min each for P0 - P4 glands, 4 min each for P5 - P14 glands, and 5 min each for P21 and older glands. Clear with xylene, 3 changes, 3 min each for P0 - P4 glands, 4 min each for P5 - P14 glands, and 5 min each for P21 and older glands. Infiltrate with molten paraffin wax, 3 changes, 7 min each for P0 - P4 glands, and 8 min twice followed by 6 min once for P5 and older glands. 
	NOTE: The optimal parameters for tissue processing can be empirically adjusted. To obtain high quality sections, insufficient or over dehydration should be avoided. The same is true for clearing the tissue using xylene.</li>
	<li>Orientation when embedding
	<ol>
		<li>On a tissue embedding console system, remove the specimen from the cassette and place it into a base mold half-filled with molten paraffin wax. Proper orientation of the embedding pituitary gland is essential for satisfying coronal sections.</li>
		<li>For P21 and adult pituitary glands, position the pituitary gland with its short axis perpendicular to the bottom surface of a base mold (the section plane). Use sphenoid bones and trigeminal nerves as anatomic landmarks for P0 - P4 and P5 - P14 pituitaries, respectively. Orient specimens with their trigeminal nerves or transversal lamina of sphenoid bones perpendicular to the bottom surface of a base mold.</li>
		<li>Gently hold the tissue in the desired position with warmed fine forceps until the wax becomes semi-solid on a cooling plate. Top up the mold with molten paraffin wax. Allow the paraffin block to cool until the wax is fully solidified. 
		NOTE: The pituitary at embryonic day 18.5 (E18.5) can be treated in the same way as the neonatal pituitary. But the pituitaries (Rathke&#39;s pouches) younger than E16.5 should be embedded with the entire head and oriented with the sagittal axis (from mouth to occiput) perpendicular to the bottom surface of a base mold.</li>
	</ol>
	</li>
	<li>Chill the paraffin block at -20 &#176;C for 10 - 20 min and then cut it into thin slices (4 &#181;m thickness is preferred) with a microtome. To get satisfactory coronal sections, fine tune the position of paraffin blocks during sectioning. Mount the tissue slices onto polylysine-coated glass slides to prevent detachment of slices in the following procedures. 
	NOTE: Alternatively, specimens can be dehydrated in 15%, 30% (W/V) sucrose solutions (in PBS) at 4 &#176;C until they sink to the bottom, embedded in cryo-embedding compound using the same orientation method, and quickly frozen on dry ice. Then cut the frozen tissue blocks into slices of 8 - 10 &#181;m using a cryostat. The morphology of cryo-sections, however, is often inferior to paraffin sections.</li>
</ol>
3. H&#38;E Staining and Immunofluorescence Labeling
<ol>
	<li>Warm the slides on a heat block at 55 &#176;C and dewax with xylene, 2 changes, 4 min each. Dehydrate the samples with 95% ethanol twice and 75% ethanol twice, 3 min each. Wash the slides with distilled water for 5 min on a shaker.</li>
	<li>For H&#38;E staining, stain the sections in alum hematoxylin solution for 6 min, rinse in running tap water for 2 min, differentiate with 0.2% ammonia water for 45 s, and re-rinse in running water for 2 min. Then dehydrate the sections with 95% ethanol for 1 min and stain with eosin for 2 min. Sequentially dehydrate, clear, and mount.</li>
	<li>Immunofluorescence labeling
	<ol>
		<li>Treat the sections with methanol containing 3% H2O2 and 0.01% NaN3 for 20 min at room temperature to inactivate endogenous peroxidases. Retrieve antigens by boiling sections in the retrieval buffer (1 mM EDTA and 1 mM Tris-HCl, pH 7.4) in a pressure cooker for 2 min. Incubate the sections with blocking buffer (5% goat serum in PBS) for 30 min at 37 &#176;C.</li>
		<li>Incubate sections with rabbit anti-growth hormone (GH) antibody (1:2,000) or rabbit anti-glial fibrillary acidic protein (GFAP) antibody (1:2,000) diluted in blocking buffer overnight at 4 &#176;C.</li>
		<li>Incubate sections with secondary antibody (goat anti-rabbit IgG-HRP, 1:300 in blocking buffer) for 35 min at 37 &#176;C.</li>
		<li>Amplify the signals using Biotinyl Tyramide (1:50 in Tris buffered saline containing 0.3% H2O2) for 15 min at room temperature, and visualize with Streptavidin-Alexa594 or 488 (1:300 in blocking buffer, 30 min, 37 &#176;C).</li>
		<li>Counterstain the nuclei with DAPI (50 mg/L in PBS) for 5 min at room temperature. 
		NOTE: It may not be necessary to amplify the signal using Tyramide Signal Amplification (TSA) system. Alternatively, a fluorescence-conjugated secondary antibody can be used to visualize the signal.</li>
	</ol>
	</li>
	<li>Acquire images using a fluorescence microscope equipped with DAPI, Texas Red, and FITC filter sets, &#215;4/0.13 to &#215;40/0.75 objectives and 5.0 megapixel camera. Use the 360, 488, and 594 nm laser wavelengths for imaging DAPI, Alexa 488-, and Alexa 594-stained sections, respectively. Optimize the laser power (0.8 was used generally) and exposure time to the desired levels for each channel. Capture the image under the control of image acquisition software and overlay the fluorescence images subsequently using image processing software.</li>
</ol>

<ol>
	<li>Fauquier, T., Lacampagne, A., Travo, P., Bauer, K., Mollard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hidden+face+of+the+anterior+pituitary.">Hidden face of the anterior pituitary.</a> Trends Endocrinol Metab. 13 (7), 304-309 (2002).</li><li>Haedo, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+pituitary+function+by+cytokines.">Regulation of pituitary function by cytokines.</a> Horm Res. 72 (5), 266-274 (2009).</li><li>Zhu, X., Gleiberman, A. S., Rosenfeld, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+pituitary+development:+signaling+and+transcriptional+networks.">Molecular physiology of pituitary development: signaling and transcriptional networks.</a> Physiol Rev. 87 (3), 933-963 (2007).</li><li>Bancalari, R. E., Gregory, L. C., McCabe, M. J., Dattani, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pituitary+gland+development:+an+update.">Pituitary gland development: an update.</a> Endocr Dev. 23, 1-15 (2012).</li><li>Kelberman, D., Rizzoti, K., Lovell-Badge, R., Robinson, I. C., Dattani, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulation+of+pituitary+gland+development+in+human+and+mouse.">Genetic regulation of pituitary gland development in human and mouse.</a> Endocr Rev. 30 (7), 790-829 (2009).</li><li>Peker, S., Kurtkaya-Yapicier, O., Kilic, T., Pamir, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsurgical+anatomy+of+the+lateral+walls+of+the+pituitary+fossa.">Microsurgical anatomy of the lateral walls of the pituitary fossa.</a> Acta Neurochir (Wien). 147 (6), 641-648 (2005).</li><li>Wolpert, S. M., Molitch, M. E., Goldman, J. A., Wood, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size,+shape,+and+appearance+of+the+normal+female+pituitary+gland.">Size, shape, and appearance of the normal female pituitary gland.</a> AJR Am J Roentgenol. 143 (2), 377-381 (1984).</li><li>Carbajo-Perez, E., Watanabe, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+proliferation+in+the+anterior+pituitary+of+the+rat+during+the+postnatal+period.">Cellular proliferation in the anterior pituitary of the rat during the postnatal period.</a> Cell Tissue Res. 261 (2), 333-338 (1990).</li><li>Nantie, L. B., Himes, A. D., Getz, D. R., Raetzman, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+in+postnatal+pituitary+expansion:+proliferation,+progenitors,+and+cell+specification.">Notch signaling in postnatal pituitary expansion: proliferation, progenitors, and cell specification.</a> Mol Endocrinol. 28 (5), 731-744 (2014).</li><li>Tzou, S. C., Landek-Salgado, M. A., Kimura, H., Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mouse+pituitary+immunogen+for+the+induction+of+experimental+autoimmune+hypophysitis.">Preparation of mouse pituitary immunogen for the induction of experimental autoimmune hypophysitis.</a> J Vis Exp. (46), (2010).</li><li>Budry, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Related+pituitary+cell+lineages+develop+into+interdigitated+3D+cell+networks.">Related pituitary cell lineages develop into interdigitated 3D cell networks.</a> Proc Natl Acad Sci U S A. 108 (30), 12515-12520 (2011).</li><li>Wilson, D. B., Wyatt, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+the+pituitary+gland+in+neonatal+little+(lit)+mutant+mice.">Histopathology of the pituitary gland in neonatal little (lit) mutant mice.</a> Histol Histopathol. 7 (3), 451-455 (1992).</li><li>Jonkers, B. W., Sterk, J. C., Wouterlood, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcardial+perfusion+fixation+of+the+CNS+by+means+of+a+compressed-air-driven+device.">Transcardial perfusion fixation of the CNS by means of a compressed-air-driven device.</a> J Neurosci Methods. 12 (2), 141-149 (1984).</li><li>Cao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZBTB20+is+required+for+anterior+pituitary+development+and+lactotrope+specification.">ZBTB20 is required for anterior pituitary development and lactotrope specification.</a> Nat Commun. 7, 11121 (2016).</li></ol>

The authors have nothing to disclose.

For developing murine pituitaries, it has been technically difficult to obtain proper coronal sections due to their tiny and fragile features and unique anatomical characteristics6,8. Some research groups thus chose mid-sagittal sections to analyze the morphology of embryonic and neonatal pituitary11,12. Though the mid-sagittal section of pituitary is also capable of showing anterior, intermediate, and po...

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis1,2. Anatomically, the pituitary gland is a &#39;&#39;two-in-one&#39;&#39; structure consisting of the neurohypophysis and the adenohypophysis. These parts have different embryonic origins and function very differently. The neurohypophysis is derived from the neural ectoderm and secretes oxytocin and antidiuretic hormone. The adenohypophysis originates from Rathke&#39;s pouch and is responsible for the release of hormones including growth hormone, prolactin, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, and melanocyte-stimulating hormone3,4,5.
The pituitary gland rests on the dorsal surface of the sphenoid bone (sella turcica) of the mouse skull and is attached to the floor of the brain by a fragile stalk. It is surrounded laterally by trigeminal nerves and anteriorly by the optic chiasm6,7. The gland has an elongated oval shape with its long axis perpendicular to that of the head. On its dorsal surface, the neurohypophysis and the adenohypophysis can easily be demarcated, with the former occupying the dorsal medial region and the latter extending laterally and ventrally. During postnatal development, the size of pituitary increases rapidly in the first month after birth8,9. Nevertheless, the mouse pituitary is still very small in size with an average weight of 1.9 mg and a long-axis diameter of ~3 mm in adult10, a long-axis diameter of 2 - 2.5 mm at postnatal day 21 (P21), and only 1 - 1.5 mm at P0.
A coronal section is preferred to display both adenohypophysis and neurohypophysis in a single slice of the mouse pituitary. However, some technical skills are required to obtain satisfying coronal sections of pituitary glands from developing mice due to its exceptionally small size and distinct anatomy. In this video article, we demonstrate how to dissect mouse pituitary glands and prepare pituitary coronal sections at different development stages.

<table>
	<tbody>
		<tr>
			<td>Tools/Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors-straight</td>
			<td>JinZhong</td>
			<td>J21010</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Fine scissors-strainght</td>
			<td>JinZhong</td>
			<td>WA1010</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Blunt forceps</td>
			<td>JinZhong</td>
			<td>JD1020</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Dumont</td>
			<td>RS-5015</td>
			<td>for isolation of the pituitary</td>
		</tr>
		<tr>
			<td>26G (0.45mm) needle</td>
			<td>HongDa</td>
			<td></td>
			<td>for transcardial perfusion</td>
		</tr>
		<tr>
			<td>Syringe (1 mL)</td>
			<td>BD</td>
			<td>300841</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Syringe (10 mL)</td>
			<td>BD</td>
			<td></td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>35mm dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Lens cleaning paper</td>
			<td>ShuangQuan</td>
			<td></td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Anatomical microscope</td>
			<td>OLYMPUS</td>
			<td>SZX-ILLB2-200</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Embedding cassette</td>
			<td>Thermo Fisher</td>
			<td>22-272423</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Tissue embedding console system</td>
			<td>KEDEE</td>
			<td>KD-BM11</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Thermo Fisher</td>
			<td>HM315R</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Superfrost-Plus slides</td>
			<td>Thermo Fisher</td>
			<td>22-037-246</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Thermo Fisher</td>
			<td>12-543</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>OLYMPUS</td>
			<td>BH2-RFCA</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Urethane</td>
			<td>BBI</td>
			<td>EB0448</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9625</td>
			<td>for PBS</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>for PBS</td>
		</tr>
		<tr>
			<td>Na2HPO4.12H2O</td>
			<td>Sigma</td>
			<td>71650</td>
			<td>for PBS</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma</td>
			<td>P2222</td>
			<td>for PBS</td>
		</tr>
		<tr>
			<td>NaNO2</td>
			<td>Sigma</td>
			<td>237213</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium Injection</td>
			<td>SPH</td>
			<td>H31022051</td>
			<td>for perfusion saline</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>SCR</td>
			<td>10009218</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>SCR</td>
			<td>10023418</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Thermo Fisher</td>
			<td>8330</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma</td>
			<td>H9627</td>
			<td>for H&#38;E staining</td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Sigma</td>
			<td>E4009</td>
			<td>for H&#38;E staining</td>
		</tr>
		<tr>
			<td>rabbit anti growth hormone (GH)</td>
			<td>National Hormone</td>
			<td></td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>antibody</td>
			<td>Pituitary Program</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse GFAP antibody</td>
			<td>Sigma</td>
			<td>G9269</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG, HRP</td>
			<td>Jackson</td>
			<td>111-035-003</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>TSA system</td>
			<td>NEN Life Science Products</td>
			<td>NEL700</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>Streptavidin, Alexa Fluor 594</td>
			<td>Thermo Fisher</td>
			<td>S32356</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>Anti-FITC Alexa Fluor 488</td>
			<td>Thermo Fisher</td>
			<td>A11090</td>
			<td>for immunostaining</td>
		</tr>
	</tbody>
</table>

dissection and coronal slice preparation of developing mouse pituitary gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

This protocol presents a method to dissect pituitary glands from developing mice. For the neonatal mouse, the whole sella regions containing the pituitary gland, the trigeminal nerves, and the underneath sphenoid bone were dissected out from the skull base. The tiny and delicate pituitary gland remained intact during the process (Figure 1A). For mice older than 5 days, the pituitary glands attached to the lateral trigeminal nerves were then isolated. The gros...

Watch this Scientific Journal Video about Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland at JoVE.com

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

dissection-coronal-slice-preparation-developing-mouse-pituitary

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

17.1K Views.  Second Military Medical University. We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

Video: Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

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Chapters in this video

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Live Confocal Imaging of Developing Arabidopsis Flowers

Dissection of Larval Zebrafish Gonadal Tissue

Dissection and Culture of Mouse Embryonic Kidney

Dissection and Staining of Drosophila Pupal Ovaries

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration